Generating a recombinant phosphothreonine-binding domain for a phosphopeptide of the human transcription factor, c-Myc

• Published in: New biotechnology Vol. 45; no. C; pp. 36 - 44
• Main Authors: Venegas, Leon A., Kall, Stefanie L., Bankole, Oluwadamilola, Lavie, Arnon, Kay, Brian K.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 25-10-2018; Elsevier Science Ltd
• Subjects: Antibodies; Antibody; Binding; c-Myc protein; Coordination compounds; Crystal structure; Domains; Engineered phosphothreonine-binding domain; Forkhead protein; Hydrophobicity; Immunoglobulins; Ligands; Localization; Myc protein; Phage display; Phages; Phosphorylation; Phosphothreonine; Polyclonal antibodies; Post-translation; Rad9 protein; Reagents; Serine; Threonine; Transcription factor c-Myc; Transcription factors; Yeast; pT + 3; Phosphorylation; pThr, pT; FBXW7; Antibody; FN3; PP2A; CDK; mAb; pAb; GSK3β; Transcription factor c-Myc; FHA1; pTBD; Phage display; CaMKII; Myc-pTBD; Myc; SCF; pSer, pS; Phosphothreonine; Engineered phosphothreonine-binding domain; ERK
• ISSN: 1871-6784; 1876-4347
• DOI: 10.1016/j.nbt.2018.05.001

•The FHA1 library yielded a reagent against a peptide from c-Myc.•The Myc-pTBD’s molecular determinants of specificity were identified.•The Myc-pTBD is comparable in specificity to commercially available antibodies.
Transcription factor c-Myc is an oncoprotein that is regulated at the post-translational level through phosphorylation of two conserved residues, Serine 62 (Ser62) and Threonine 58 (Thr58). A highly specific tool capable of recognizing Myc via pThr58 is needed to monitor activation and localization. Through phage display, we have isolated 10 engineered Forkhead-associated (FHA) domains that selectively bind to a phosphothreonine (pThr)-containing peptide (53-FELLPpTPPLSPS-64) segment of human c-Myc. One domain variant was observed to bind to the Myc-pThr58 peptide with a KD value of 800 nM and had >1000-fold discrimination between the phosphorylated and non-phosphorylated peptide. The crystal structure of the engineered FHA Myc-pThr-binding domain (Myc-pTBD) was solved in complex with its cognate ligand. The Myc-pTBD was observed to be structurally similar to the yeast Rad9 FHA1 domain, except that its β4-β5 and β10-β11 loops form a hydrophobic pocket to facilitate the interaction between the domain and the peptide ligand. The Myc-pTBD’s specificity for its cognate ligand was demonstrated to be on a par with 3 commercial polyclonal antibodies, suggesting that this recombinant reagent is a viable alternative to antibodies for monitoring Myc regulation.